This proposal has three specific, short term aims: 1.) cloning C. neoformans genes likely to be important for pathogenesis; 2.) constructing "knock-out" mutations in these genes; nd 3.) testing the resulting C. neoformans mutants in experimental infections. The goal of these experiments is to identify genes-mutants that are avirulent/non- pathogenic in experimental infections and thereby identify good potential drug targets. The primary focus is on a specific subset of amino acid and vitamin biosynthetic genes. The reason for focusing on these pathways is that many auxotrophs are avirulent. The choice of genes in these pathways is dictated by (i) their absence in humans and (ii) the highly deleterious in vitro phenotypes of specific mutants. The secondary focus is on gene products that are required for growth only at high temperature. The reason for focusing on such gene products is that the ability to growth at high temperatures is essential for fungal virulence-pathogenesis. This proposal is health related because (i) it deals with C. neoformans, one of the most serious of the pathogenic fungi, and (ii) it is focused on the identification of antifungal drug targets. The currently available antifungal drugs are few (compared to antibacterials) in number, are less effective than clinicians would like, and tend to have severe side effects. Therefore, there is a great need to develop new antifungals. The goal of this proposal is to genetically identify new antifungal drug targets. Once specific new targets have been identified it will be possible to focus attention on these gene products and use novel technologies, such as combinatorial chemistry, to develop small molecule inhibitors. Such collaborations are possible here at Duke (see attached letters). This proposal has three broad long term/future goals: 1.) to examine more genes/mutants to get a global picture of C. neoformans pathogenesis; 2.) to apply this approach to other pathogenic fungi; and 3.) to develop small molecule inhibitors of gene products that are essential for pathogenesis.